Cloning and characterization of thermostable endoglucanase (Cel8Y) from the hyperthermophilic Aquifex aeolicus VF5

Aquifex aeolicus is the hyperthermophilic bacterium known, with growth-temperature maxima near 95 degreesC. The cel8Y gene, encoding a thermostable endoglucanase (Cel8Y) from Aquifex aeolicus VF5, was cloned into a vector for expression and expressed in Escherichia coli XL1-Blue. A clone of 1.7 kb fragment containing endoglucanase activity, designated pKYCY100, was sequenced and found to contain an ORF of 978 bp encoding a protein of 325 amino acid residues, with a calculated molecular mass of 38,831 Da. This endoglucanase was designated cel8Y gene. The endoglucanase has an 18-amino-acid signal peptide but not cellulose-binding domain. The endoglucanase of A. aeolicus VF5 had significant amino acid sequence similarities with endoglucanases from glycosyl hydrolase family 8. The predicted amino acid sequence of the Cel8Y protein was similar to that of CMCase of Cellulomonas uda, BcsC of Escherichia coli, CelY of Erwinia chrysanthemi, and CMCase of Acetobacter xylinum. The molecular mass of Cel8Y was calculated to be 36,750 Da, which is consistent with the value obtained from result of CMC-SDS-PAGE of the purified enzyme. Cel8Y was thermostable, exhibiting maximal activity at 80 degreesC and pH optima of 7.0 and with half-lives of 2 h at 100 degreesC, 4 h at 90 degreesC. (C) 2000 Academic Press.